Studies demonstrate that between 25-50% of menopausal women report vulvovaginal symptoms including vaginal dryness, irritation, burning, itching, and dyspareunia, all of which severely affect quality of life. As women approach menopause, decreased estrogen levels are accompanied by changes in the vagina including decreased glycogen accumulation in the epithelium, increased pH, and deterioration of vaginal tissues, which lead to symptoms of vaginal discomfort. Hormone therapy is the primary treatment for vulvovaginal symptoms in menopausal women, however it is contraindicated in some women. Non-hormonal therapeutic approaches for improving symptoms and quality of life in postmenopausal women have not been well studied. The vaginal microbial communities (termed the vaginal microbiota) play a critical role in protecting the female genital tract from disease; however, surprisingly little is known about the composition of vaginal bacteria across the different stages of menopausal transition, how they differ between individuals, the correlation with vaginal immunologic microenvironment, or the associations with gynecologic complaints and sexual functioning. Between 2009-2012, 885 women aged 35-60 years were enrolled in a prospective study of menopause transition and followed every six months for two years (R01-CA123467). Taking advantage of that resource, the proposed study will utilize 2500 longitudinal vaginal samples stored in a freezer archive to address the following three specific aims: (1) To describe the changes in the vaginal microbiota by age and stage of menopause over a 2-year period, (2) To evaluate the association between the vaginal microbiota and vulvovaginal complaints and (3) To assess the correlation between the vaginal immunologic microenvironment with the microbiota. Participants were categorized according to menopausal categories by the Stages of Reproductive Aging Workshop (STRAW) guidelines. The vaginal microbiota will be characterized using 16S rRNA gene analysis amplified from whole genomic DNA isolated from clinician-collected mid-vaginal swabs. Cervical secretions will be used to quantify concentrations of 27 different immune markers (cytokines, chemokines, and growth factors) using multiplexed bead-based immunoassays with total protein adjustment. By bringing together multidisciplinary expertise in bioinformatics, epidemiology, genomics, gynecology, microbiology and statistics, this study will be able to provide critical descriptive data on (1) differences in the composition and stability of the vaginal microbiome by menopausal stage, (2) association between microbial changes and presence of vulvovaginal complaints, and (3) the possible correlation between vulvovaginal microbial and immunologic microenvironmental changes during the menopausal transition. These data will be used to evaluate the potential for non-hormonal, anti-inflammatory, or probiotic interventions for the treatment of common vulvovaginal complaints in peri- and postmenopausal women.